GSM6771484: Plasmablast v2D9 - ADT; Homo sapiens; RNA-Seq
B cells of three vaccinees were isolated from thawed cryopreserved peripheral blood mononuclear cells (PBMC) by negative magnetic bead-based selection using a B cell enrichment cocktail (StemCell Technologies). The isolated B cells were stained with PE-Cy7-conjugated anti-human CD19 (ThermoFisher, cat# 25-0198-42) and APC-H7-conjugated anti-human CD20 (BD Biosciences, cat# 560734). SARS-CoV-2-specific B cells were identified using biotinylated spike protein (S-2P) tetramerized with PE-Cy5.5-labeled streptavidin (SA) (ThermoFisher, cat# SA1018) at a molar ratio of 4:1. Three B cell populations were sorted, (1) CD20+S-2P+ B cell, (2) CD20+S-2P-  B cell, and (3) CD20- B cell (plasmablast) populations, using a BD FACSAria Fusion instrument (BD Biosciences). Isolated B cells from different donors were pooled evenly before cell sorting. Cells from each donor were demultiplexed in silico using donor-specific single nucleotide polymorphism (SNP) information collected during data analysis. Multiple cell pools were prepared, such that cells from the same donors but different time points were in different pools. After cell sorting, cell pools from v2D28 and v2M6 timepoints were Fc blocked (Human TruStain FcX, BioLegend), CITE-seq and hashtag stained with custom TotalSeq-C cocktail antibodies, (including RBD and S1 oligo-tagged tetramers, and washed 3x with staining buffer (2% BSA in PBS). Due to the lower frequency of S-2P+ B cells at earlier time points, pools from v1D14 to v2D14 timepoints were CITE-seq and hashtag (HTO) stained before cell sorting to avoid cell loss during washes. No RBD and S1 oligo-tagged tetramers could be added due to technical reasons. Final cell pools were suspended in 0.04% BSA in PBS and then counted before being immediately processed by the single cell partitioning step. Cell pools were mixed with a reverse transcription (RT) mix and partitioned into single cell Gel-Bead in Emulsion (GEM) using the 10x Chromium Single Cell Immune Profiling Next GEM v2 Chemistry Kit (10x Genomics, Pleasanton, CA). The RT step was conducted using a C1000 touch Thermo Cycler (BioRad, Hercules, CA). Single-cell gene expression (GEX), cell surface protein (ADT) and B cell receptor (BCR) libraries were prepared as instructed by 10x Genomics Single Cell Immune Profiling kit v.2 user guide. A total of 84 libraries were quality control tested using a TapeStation 4200 (Agilent, Santa Clara, CA) and quantified using the KAPA Library Quantification kit (Roche, Wilmington, MA). These libraries were then pooled and sequenced on Illumina NovaSeq platform (Illumina, San Diego, CA) using the sequencing parameters recommended by the 10x Genomics user guide. The minimum sequencing depths were 50,000 read pairs/cell for the GEX library, 5,000 read pairs/cell for the BCR library, and 5,000 read pairs/cell for the ADT library. Sequencing saturation of the libraries was 80% for the GEX and 10% for the BCR and ADT libraries.